Beta-3 adrenergic receptor activation via exercise or CL316,243 (CL) induces white adipose tissue (WAT) browning, improves glucose tolerance, and reduces visceral adiposity. Our aim was to determine if sex or adipose tissue depot differences exist in response to CL. Daily CL injections were administered to diet-induced obese male and female mice for two weeks, creating four groups: male control, male CL, female control, and female CL. These groups were compared to determine the main and interaction effects of sex (S), CL treatment (T), and WAT depot (D). Glucose tolerance, body composition, and energy intake and expenditure were assessed, along with perigonadal (PGAT) and subcutaneous (SQAT) WAT gene and protein expression. CL consistently improved glucose tolerance and body composition. Female PGAT had greater protein expression of the mitochondrial uncoupling protein 1, while UCP1 (S, p < 0.001) was more responsive to CL in increasing UCP1 (S×T, p = 0.011) and the mitochondrial biogenesis induction protein, PPARγ coactivator 1α (PGC1α) (S×T, p = 0.026). Females also displayed greater mitochondrial OXPHOS (S, p < 0.05) and adiponectin protein content (S, p < 0.05). On the other hand, male SQAT was more responsive to CL in increasing protein levels of PGC1α (S×T, p = 0.046) and adiponectin (S, p < 0.05). In both depots and in both sexes, CL significantly increased estrogen receptor beta (ERβ) and glucose-related protein 75 (GRP75) protein content (T, p < 0.05). Thus, CL improves systemic and adipose tissue-specific metabolism in both sexes; however, sex differences exist in the WAT-specific effects of CL. Furthermore, across sexes and depots, CL affects estrogen signaling by upregulating ERβ.

Estrogen receptor  (ERb), one of the two major estrogen receptors, acts via genomic and non-genomic signaling pathways to affect many metabolic functions, including mitochondrial biogenesis and respiration. This study assessed the effect of ERb classical genomic activity on adipocyte-specific and systemic metabolic responses to wheel running exercise in a rodent model of menopause. Female mice lacking the ERb DNA-binding domain (ERbDBDKO, N = 20) and wild-type (WT, N = 21) littermate controls were fed high-fat diet (HFD), ovariectomized (OVX), and randomized to control (no running wheel) and exercise (running wheel access) groups and were followed for eight weeks. Wheel running did not confer protection against metabolic dysfunction associated with HFD+OVX in either ERbDBDKO or WT mice, despite increased energy expenditure. Unexpectedly, in the ERbDBDKO group, wheel running increased fasting insulin and surrogate measures of insulin resistance, and modestly increased adipose tissue inflammatory gene expression (P<0.05). These changes were not accompanied by significant changes in adipocyte mitochondrial respiration. It was demonstrated for the first time, that female WT OVX mice do experience exercise-induced browning of white adipose tissue, indicated by a robust increase in uncoupling protein 1 (UCP1) (P<0.05). However, KO mice were completely resistant to this effect, indicating that full ERb genomic activity is required for exercise-induced browning. The inability to upregulate UCP1 with exercise following OVX may have resulted in the increased insulin resistance observed in KO mice, a hypothesis requiring further investigation.

White adipose tissue (WAT) dysfunction independently predicts cardiometabolic disease, yet there is a lack of effective adipocyte-targeting therapeutics. B3AR agonists enhance adipocyte mitochondrial function and hold potential in this regard. Based on enhanced sensitivity to B3AR-mediated browning in estrogen receptor (ER)alpha-null mice, we hypothesized that ERβ may enhance the WAT response to the B3AR ligand, CL316,243 (CL).Methods: Male and female wild-type (WT) and ERβ DNA binding domain knock-out (ERβDBDKO) mice fed high-fat diet (HFD) to induce obesity were administered CL (1 mg/kg) daily for 2 weeks. Systemic physiological assessments of body composition (EchoMRI), bioenergetics (metabolic chambers), adipocyte mitochondrial respiration (oroboros) and glucose tolerance were performed, alongside perigonadal (PGAT), subcutaneous (SQAT) and brown adipose tissue (BAT) protein expression assessment (Western blot). Mechanisms were tested in vitro using primary adipocytes isolated from WT mice, and from Esr2-floxed mice in which ERβ was knocked down. Statistical analyses were performed using 2 × 2 analysis of variance (ANOVA) for main effects of genotype (G) and treatment (T), as well as GxT interactions; t-tests were used to determine differences between in vitro treatment conditions (SPSS V24).Results: There were no genotype differences in HFD-induced obesity or systemic rescue effects of CL, yet ERβDBDKO females were more sensitive to CL-induced increases in energy expenditure and WAT UCP1 induction (GxT, p &lt; 0.05), which coincided with greater WAT B3AR protein content among the KO (G, p &lt; 0.05). Among males, who were more insulin resistant to begin with (no genotype differences before treatment), tended to be more sensitive to CL-mediated reduction in insulin resistance. With sexes combined, basal WAT mitochondrial respiration trended toward being lower in the ERβDBDKO mice, but this was completely rescued by CL (p &lt; 0.05). Confirming prior work, CL increased adipose tissue ERβ protein (T, p &lt; 0.05, all), an effect that was enhanced in WAT and BAT the female KO (GxT, p &lt; 0.01). In vitro experiments indicated that an inhibitor of ERβ genomic function (PHTPP) synergized with CL to further increase UCP1 mRNA (p = 0.043), whereas full ERβ protein was required for UCP1 expression (p = 0.042).Conclusion: Full ERβ activity appears requisite and stimulatory for UCP1 expression via a mechanism involving non-classical ERβ signaling. This novel discovery about the role of ERβ in adipocyte metabolism may have important clinical applications.

In vivo, ERb activation protects against obesity and white adipose tissue (WAT) dysfunction. Mechanisms are unknown, but we hypothesize that it involves adipocyte‐specific mitochondrial activity via induction of OXPHOS and UCP1– proteins that characterize WAT “browning,” a process sufficient to rescue metabolic dysfunction. In cancer cells, increasing mitochondrial ERb increases OXPHOS transcription and mitochondrial activity via a mechanism requiring mitochondrial transporter, GRP75. We sought to determine whether this mechanism applies to adipocytes in vitro, and the potential in vivo implications. In wild type (WT) mice, the adipocyte‐specific B3AR ligand, CL316,243 (CL) induced WAT UCP1 and ERb protein (Western blot), and GRP75 mRNA (qPCR) (all n=10/grp, P<0.05). In primary adipocytes from WT mice (n=3/treatment), CL increased UCP1 (~2‐fold; P<0.05) and ERb protein (~4‐fold; trend, P=0.097). In vivo, we show that adipocytes harvested from mutant mice missing ERb DNA binding domain (ERbDBDKO) display suppressed mitochondrial O2 consumption (via Oroboros; n=6–8/grp, P<0.05) and lower OXPHOS protein expression (P<0.05). We next tested whether ERbDBDKO mice would have impaired exercise‐induced WAT browning. Remarkably, unlike WT (~3‐fold UCP1 increase; P<0.05), ERbDBDKO mice were nonresponsive to exercise induction of UCP1 protein in WAT. Taken together, our data support a critical role for adipocyte ERb in mediating lipolysis driven mitochondrial responses. We hypothesize that the mechanism involves GRP75. Identifying the mechanism by which ERb enhances adipocyte mitochondrial activity has enormous potential to treat a spectrum of metabolic diseases.